The overall objective of this project is to understand the mechanism of the pathogenesis and oncogenesis of hepatitis C virus (HCV). HCV is one of the major causes of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). It is also associated with B cell oligoclonal proliferation and non-Hodgkin's B cell lymphoma in certain geographical regions. The study of HCV has been hampered by the lack of an efficient tissue culture system for propagating HCV. Our laboratory recently established a B cell culture system for HCV infection, which allows the in vitro infection of culture cells and persistent production of HCV particles. Our preliminary findings indicate that HCV infection induces double-strand DNA breaks and apoptosis and enhances mutation frequency of cellular genes, suggesting that HCV induces a mutator phenotype. Our system thus opens up a new and unique approach for studying HCV biology in the context of complete viral infection and replication. Our specific aims for this project are: SPECIFIC AIM 1. Characterization of the biological properties of HCV-infected B cells. (a) Apoptosis associated with HCV infections, In particular, we will study the role of nitric oxide in the induction of double-strand DNA breaks, and apoptosis and its effect on HCV replication will be studied. We will also study, the mechanism of iNOS induction and the role of the active oxygen species in HCV infection. (b) The mutagenic properties of HCV infection. We will examine whether HCV infection increases the mutation frequency of cellular DNAs, causes chromosomal breaks and/or inhibits DNA repair mechanisms. (c) The co-evolution of the immunoglobulin gene and viral RNA sequences during in vitro passages. This study will contribute to the understanding of whether B cell properties are affected by HCV infection. specific aim 2. Identification of the viral gene products responsible for the various biological properties studied in Specific Aim 1. To understand the mechanism of the biological changes induced by HCV. we will examine the ability of the individual HCV gene products to induce the phenomena described in Specific Aim 1. Individual viral genes will be constructed and expressed in B cells and hepatocyte cell lines. The various parameters of apoptosis and DNA mutations will be examined. These include iNOS induction, double-strand DNA breaks, and induction of error-prone DNA polymerases. The possible involvement of extracellular E2 protein in causing these effects will also be examined.